The serotonin transporter (5-HTT) regulates serotonergic neurotransmission by high affinity uptake of released serotonin. It is a key site of action for many psychotherapeutic and addictive drugs. The level of 5-HTT expression varies in humans according to whether an individual carries the short (s) variant of the 5-HTT promoter region polymorphism (5-HTTLPR). Carriers of the s allele express ~50% fewer 5-HTTs than those homozygous for the long (/) allele. Carriers of the s allele appear to be more prone to a variety of psychiatric disorders, including depression and drug abuse, and are often resistant to treatment with selective serotonin reuptake inhibitors (SSRIs) compared to individuals homozygous for the / allele. To study the neurochemical underpinnings of the relationship between 5-HTT genotype and drug effect, we have made use of mice with a null mutation of the 5-HTT. Heterozygote 5-HTT mutants resemble carriers of the s allele in that they express 50% fewer 5-HTTs and clear serotonin from extracellular fluid more slowly than wild-type mice. The null mutants lack 5-HTTs. One of our most intriguing findings is that 5-HTT mutant mice are more sensitive to the serotonin clearance inhibiting effect of "Ecstasy" (MDMA) and alcohol where greatest inhibition of serotonin clearance occurs in null mutants. Thus, these drugs inhibit serotonin clearance but via a 5-HTT independent mechanism. Importantly, this mechanism has presumably undergone compensatory upregulation in 5-HTT mutants. The studies proposed here seek to identify these alternative mechanisms for serotonin transport. It is known that the dopamine and norepinephrine transporters take up serotonin, but preliminary data indicate that at least in the CA3 region of hippocampus these transporters do not play a significant role in serotonin clearance in 5-HTT mutant mice. By contrast, blockers of the organic cation (OCT) and proline (PROT) transporters profoundly inhibit serotonin clearance in 5-HTT mutant mice. Here we will determine the relationship between genetically defined deficiencies in 5-HTT expression and (1) OCT and PROT expression, (2) OCT and PROT function and (3) behavior to assess changes in effectiveness of SSRIs and blockers of the OCT and PROT in preclinical measures for antidepressant efficacy and treatment of alcoholism. We will also investigate OCT and PROT as sites of action for antidepressant and abused drugs and how their efficacy varies with 5-HTT genotype. In addition to increasing our fundamental understanding of serotonergic neurotransmission, these studies may lead to novel treatments for affective and addictive disorders that are better tailored to the individual. The clinical implications for upregulation of alternative mechanisms for serotonin uptake when 5-HTT expression is compromised are far reaching. That is, drugs targeted at OCTs or PROT may represent first line or adjunctive therapies for individuals who do not respond well to treatment with SSRIs.